Atomic structures of membrane proteins are playing a major role in understanding the mechanisms of action and deriving fundamental principles for these systems. Crystallography is the dominant method for obtaining membrane protein structures at atomic and near atomic resolutions, single-wavelength anomalous diffraction (SAD) predominates for de novo structure determination, which is essential in many instancs. Recently x-ray free electron lasers for crystallography and direct electron detectors for single-particle cryo electron microscopy (cryo-EM) emerging as technologies for solving challenging structures of membrane proteins and their complexes. Nevertheless, bottlenecks remain including weak anomalous signals, micron-sized crystals and radiation damage in crystallography and atomic interpretation of electron density maps from cryo-EM. We propose to address these bottleneck challenges in four sub-projects. Aim 1 (TR&D 4.1) focuses on the extraction of signals from weak anomalous scatterers with the aim of making native-SAD analysis robust even for low-resolution membrane protein structures. Aim 2 (TR&D 4.2) relates to challenges in the manipulation and analysis of microcrystals, including the development of new methods for mounting microcrystals presentation in microdiffraction experiments and methods of analysis for optimal synthesis of complete data sets from the partial data of many crystals. Aim 3 (TR&D 4.3) is concerned with the mitigation of radiation damage, which is exacerbated in measurements from microcrystals as needed for optimal native-SAD analyses. Aim 4 (TR&D 4.4) will address the optimized fitting of atomic models to cryo-EM maps. We will innovate methods for identifying specific residues types in such maps and methods for a sorted classification of ligand-binding populations for optimized placement of the ligands and associated conformational changes.